Members of the cytochrome P-450 (CYP) family of hemoproteins are critical in the oxidative metabolism of a wide variety of endogenous substances and xenobiotics, including various carcinogens and toxins (Nebert et al, Ann. Rev. Biochem. 56:945-993 (1987)). In man, the CYP3A4 monooxygenase plays a major role in the biotransformation of drugs due to its abundance in liver and intestine and its broad substrate specificity. CYP3A4 catalyzes the metabolism of >60% of all drugs that are in use including steroids, immunosuppressive agents, imidazole antimycotics, and macrolide antibiotics (Maurel, P. in Cytochromes P450: metabolic and toxicological aspects (ed. Ioannides, C.) 241-270 (CRC Press, Inc., Boca Raton, Fla., 1996).
Expression of the CYP3A4 gene is markedly induced both in vivo and in primary hepatocytes in response to treatment with a variety of compounds. Many of the most efficacious inducers of CYP3A4 expression are commonly used drugs such as the glucocorticoid dexamethasone, the antibiotic rifampicin, the antimycotic clotrimazole, and the hypocholesterolemic agent lovastatin (Maurel, P. in Cytochromes P450: metabolic and toxicological aspects (ed. Ioannides, C.) 241-270 (CRC Press, Inc., Boca Raton, Fla., 1996), Guzelian, P. S. in Microsomes and Drug Oxidations (eds. Miners, J. O., Birkett, D. J., Drew, R. & McManus, M.) 148-155 (Taylor and Francis, London, 1988). The inducibility of CYP3A4 expression levels coupled with the broad substrate specificity of the CYP3A4 protein represent the basis for many drug interactions in patients undergoing combination drug therapy. While attempts have been made to develop in vivo and in vitro assays with which to profile the effects of compounds on CYP3A expression levels, these efforts have been hampered by species-specific effects that have limited the utility of using animals and their tissues for testing purposes. Thus, analysis of the effects of new compounds on CYP3A4 gene expression has been largely restricted to laborious assays involving human liver tissue.
Recently, efforts have been directed at understanding the molecular basis for the induction of CYP3A4 gene expression. The CYP3A4 promoter has been cloned and a 20 bp region residing approximately 150 bp upstream of the transcription initiation site shown to confer responsiveness to dexamethasone and rifampicin (Hashimoto et al, Eur. J. Biochem. 218:585-595 (1993), Barwick et al, Molec. Pharmacol. 50:10-16 (1996)). This region contains two copies of the AG(G/T)TCA motif recognized by members of the nuclear receptor superfamily, suggesting that a nuclear receptor might be responsible for mediating at least some of the effects of the chemical inducers of CYP3A4 expression. However, prior to the present invention, proteins that bind to this response element had not been characterized.
The present invention is based on the identification of a novel orphan nuclear receptor that binds to a response element in the CYP3A4 promoter and that is activated by a range of compounds known to induce CYP3A4 expression. The identification of this receptor makes possible assays that can be used to establish whether drugs will interact in vivo.